§ 436.305 - Thin layer chromatographic identity test for hetacillin.

(a) Equipment—(1) Chromatography tank. A rectangular tank, approximately 9 × 9 × 3.5 inches with a glass solvent trough on the bottom.

(2) Plates. Use 20 × 20 centimeter thin layer chromatography plates coated with Silica Gel G or equivalent to a thickness of 250 microns.

(b) Developing solvent. Mix 650 milliliters acetone with 100 milliliters distilled water, 100 milliliters benzene, and 25 milliliters acetic acid.

(c) Spray solution. Dissolve 300 milligrams of ninhydrin in 100 milliliters of ethanol.

(d) Preparation of spotting solutions—(1) Sample solution. Use the sample solution prepared as described in the section for the particular product to be tested.

(2) Reference solutions. Prepare a solution containing 10 milligrams of an authentic hetacillin sample per milliliter in a 4:1 solution of acetone and 0.1N hydrochloric acid, and a solution of ampicillin standard at 1 mg/ml in the same solvent.

(e) Procedure. Spot a plate as follows: Apply approximately 10 microliters of the sample solution, 1 μ l, of the reference hetacillin solution, and 1 μ l, of the ampicillin reference solution on a line 1.5 centimeters from the base of the silica gel plate and at intervals of not less than 2.0 centimeters. Pour developing solvent into the glass trough in the bottom of the chromatography tank. After all spots are thoroughly dry, place the silica gel plate directly into the glass trough of the chromatography tank. Cover and seal the tank. Allow the solvent front to travel about 11.5 centimeters from the bottom of the plate, remove the plate from the tank, and allow to air dry. Apply the spray solution (do not saturate) and place immediately into an oven maintained at 90° C. Heat 15 minutes.

(f) Evaluation. Measure the distance the solvent front traveled from the starting line and the distance the spots are from the starting line. Calculate the Rf value by dividing the latter by the former. The sample and standard should have spots of corresponding Rf values.